Use Of Metagenomic Approaches To Isolate Lipolytic Genes From Environmental Sample

The fact that99%of microbes in the environment cannot be cultured under laboratory conditions has severely inhibited the full exploitation of microbial genetic resources. Metagenimics, however, independent on the microbial culture, provides a valuable access to the genetic resources. In this study, a plasmid metagenomic library derived from soil which composed of19500genes and a Fosmid metagenomic library of20400genes derived from contaminated water have been established. Then the tributyrin plate-based screening method has been utilized; through the method, a positive clone has been obtained from the soil plasmid metagenomic library and then sequenced and identified as the esterase gene estP2K which encodes a protein of224amino acids. Six positive clones were obtained from the Fosmid metagenomic library and then the fosmids were extracted and digested to form a subclone library from which the esterase gene estF4K of1188bp was gained. The sequences of the amino acids indicated that the similarity between EstP2K and Neisseria elongata subsp is just37%; and EstF4K possesses SMTK domain of the esterase/lipase family. The similarity between β-lactamase from the unculturable microbes in the Genbank and the sequences of amino acids of EstF4K is about83%. The respectively enzymologic analysis of EstP2K and EstF4K after the heterologous protein expression has shown that EstP2K is much more active to the p-nitrophenyl (pNP) esters of mid-length carbon chain fatty acids than to pNP esters of the short-chain fatty acids. The optimum temperature and pH for EstP2K are at45℃and7.5and Mg2+can significantly increase its enzyme activity. EstF4K has shown a preference to the pNP esters of the short chain fatty acides and the optimum temperature and optimum pH are at50℃and8.0.